The present invention relates generally to modification of plant fatty acid composition by expression of a plant xcex949 acyl-CoA desaturase, particularly selective and preferential increases in the ratio of oleic acid to stearic acid.
Lipids are essential in the composition of all plant cells. Although plant lipids cover a wide range of compounds, the majority of lipids are derived from two important metabolic pathways, the fatty acid biosynthetic pathway and the glycerolipid biosynthetic pathway. Plants naturally produce an assortment of fatty acids which they incorporate into a wide assortment of lipids which perform different functions. Polar glycerolipids (phospholipids and glycolipids), for example, contain two fatty acids attached to both sn-1 and sn-2 positions of the glycerol backbone and a polar headgroup attached to the sn-3 position. Polar glycerolipids play an essential role in cell membrane structure and function. Triacylglycerols, on the other hand, have all three positions of the glycerol backbone esterified with fatty acids and are the major storage lipids in oil-producing plant tissues, such as in plant seeds, and are usually known as plant oils.
The specific properties of a plant oil are dependent on the fatty acid composition of the oil, which in turn affects the nutritional quality of the oil. The health value of high levels of monounsaturates, particularly oleic acid, as the major dietary fat constituent has been established by recent studies. For example, canola oil, which typically contains at least 60% oleic acid (c18:1, xcex949), has been proven effective in lowering cholesterol in human blood. It has also been shown, however, that high levels of all monounsaturated fatty acids are not necessarily beneficial. For example, it has been suggested that palmitoleic acid (c16:1, xcex949) may have certain health disadvantages, such as behaving as a saturated fatty acid in its effect on cholesterol (Nestel et al., 1994, J Lipid Res 35(4):656-662) effecting atrioventicular conduction in the heart (Dhein et al, 1999, Br. J. Pharmacol 128(7) 1375-1384) and correlating with high blood pressure in men at high risk of coronary heart disease (Simon et al., Hypertension Feb. 27, 1996 (2):303-7). As a result, because of these medical and nutritional effects, there is an interest in lowering the level of saturated fatty acids in plant oils beyond certain limits (the limit of allowable saturated fatty acid proportions in canola oil, for example, is 7%).
The fatty acid composition of plant oils is determined both by the genotype of the plant and the plant""s response to environmental factors such as light, temperature and moisture. Genetic modification by plant breeding or genetic engineering may be used to modify fatty acid metabolic pathways and thereby modify plant oil composition.
In plants, fatty acids are generally synthesized in the plastid or chloroplast by the FAS system in which the elongating chain is generally esterified to acyl-carrier protein (ACP) as palmitic acid (16:0) and stearic acid (18:0) esterified to ACP (i.e., 16:0-ACP and 18:0-ACP, respectively). A known soluble plant stearoyl-ACP xcex949 desaturase enzyme is located in the chloroplast where it is understood to catalyze the conversion of stearoyl-ACP (18:0-ACP) to oleoyl-ACP (18:1-ACP). These acyl-ACPs may either be used for glycerolipid synthesis in the chloroplast or transported out of chloroplast into the cytoplasm as acyl-CoAs. It is generally believed that the stearoyl-ACP xcex949 enzyme is the only soluble plant desaturase, so that palmitic acid and stearic acid exported from the chloroplast will not undergo further desaturation. Therefore, the level of saturation is largely determined by the amount of saturated fatty acids exported out of the chloroplast and into the cytoplasm.
This situation in plants is in contrast to that known for yeast and mammalian acyl-CoA xcex949 desaturases, which use fatty acids esterified to CoA as substrates, and desaturate both the saturated fatty acids palmitic acid and stearic acid. Mammalian and yeast acyl-CoA xcex949 desaturases have been used to modify levels of saturated fatty acids in plant tissues (U.S. Pat. Nos. 5,866,789 and 5,777,201) and have been shown to result in increased levels of monounsaturated fatty acids, including both oleic and palmitoleic fatty acids, and decreased levels of saturated fatty acids in plant oils. Recently, two genes homologous to the mammalian and yeast acyl-CoA desaturases were isolated from Arabidopsis, ADS1 and ADS2 respectively (Fukuchi-Mizutani et al. (1998) Plant Cell Physiol. 39:247-253). ADS1 and ADS2 share 76% amino acid sequence identity and it has been speculated that these two genes are xcex949 fatty acid desaturases. The Genbank database accession for the ADS1 protein and nucleic acid sequences is D88536, which sets out the sequences as follows:
(SEQ ID NO: 9)
(SEQ ID NO: 1)
The Genbank database accession for the ADS2 protein and nucleic acid sequences is D88537, which sets out the sequences as follows:
(SEQ ID NO: 10)
(SEQ ID NO: 2)
In view of the influence on health and nutrition, there is a continuing need for methods for modifying the fatty acid composition of plant parts, such as plant oils.
In various embodiments, the invention provides for the use of a ADS1 or ADS2 xcex949 fatty acid desaturase to selectively increase the relative proportion of oleic acid in the fatty acid of a plant part, such as in the oil of a mature seed. In some embodiments, the proportion of oleic acid may be increased preferentially, without a corresponding or proportional increase in palmitoleic acid.
In one aspect, the invention provides a method for modifying the fatty acid content of a plant part, such as an oil-producing plant tissue. In one aspect, the method may comprise the step of introducing a DNA sequence encoding an ADS1 or ADS2 xcex949 fatty acid desaturase into a plant cell of the plant, or an ancestor of the plant, to produce a genetically modified plant comprising the DNA sequence. The genetically modified plant may be maintained under conditions so that the DNA sequence encoding the ADS1 or ADS2 xcex949 fatty acid desaturase is expressed. The ratio of oleic acid (18:1) to stearic acid (18:0) may be increased by a selected value, such as by at least 20%, in a part of the genetically modified plant, compared to a corresponding part of a non-modified plant. The ratio of palmitoleic acid (16:1) to palmitic acid (16:0) in the part of the plant may also be decreased or remain unchanged or increase by an amount less than a selected value, such as by no more than 20%, compared to the corresponding part of the non-modified plant.
In one aspect, the invention provides genetically modified plants comprising a heterologous DNA sequence encoding an ADS1 or ADS2 xcex949 fatty acid desaturase. The DNA sequence encoding the ADS1 or ADS2 xcex949 fatty acid desaturase may be expressed so that the ratio of oleic acid (18:1) to stearic acid (18:0) is increased, for example it may be increased by at least 20%, in a part of the genetically modified plant, compared to a corresponding part of a non-modified plant. The ratio of palmitoleic acid (16:1) to palmitic acid (16:0) may also be decreased or remain unchanged or increased by no more than a selected value, such as 20%, in the part of the genetically modified plant.
In another aspect, the invention provides plant parts, such as an oil obtainable from an oil-producing plant tissue (such as from seeds), wherein the plant part has an increased ratio of oleic acid to stearic acid.